Human 3 relaxin

ABSTRACT

Human H3 preprorelaxin, human H3 prorelaxin, human H3 relaxin, human relaxin analogues having a modified A chain and/or a modified B chain are described. Also described are nucleic acid sequences encoded human H3 preprorelaxin, human H3 prorelaxin, human H3 relaxin, human relaxin analogues. Also described are methods for the treatment of conditions responsive to administration of H3 relaxin or analogues thereof.

FIELD OF THE INVENTION

This invention relates to human 3 relaxin (hereafter referred to as “H3relaxin”). More specifically, the invention relates to H3 relaxin, pro-and prepro-H3 relaxin, the individual peptide chains which comprisethese sequences, analogues of H3 relaxin, compositions includingpharmaceutical compositions, as well as therapeutic uses and methods oftreatment. Further, the invention relates to nucleic acids encoding H3relaxin, H3 pro- and prepro-relaxin, H3 relaxin analogues, andindividual peptide chains which comprise these sequences.

BACKGROUND OF THE INVENTION

Pioneering work by Hisaw 1926 first suggested an important role for thepeptide hormone relaxin in animals through its effect in dilating thepubic symphsis, thus facilitating the birth process. Relaxin issynthesised in the corpora lutea of ovaries during pregnancy, and isreleased into the blood stream prior to parturition. The availability ofovarian tissue has enabled the isolation and amino acid sequencedetermination of relaxin from the pig (James et al (1977), Nature, 267,554-546), the rat (John et al (1981) Endocrinology 108, 726-729), andthe shark (Schwabe et al (1982) Ann. N.Y. Acad. Sci. 380, 6-12).

Relaxin genes and the encoded relaxin polypeptides have been identifiedin many species including man, pig, rat, sheep and shark. In all thesespecies only one relaxin gene has been characterised in mammals, -withthe exception of the human and higher primates where two separate geneshave been described. The separate human genes were identified by thepresent applicant and designated H1 (Hudson et al (1983) Nature 301,628-631) and H2 (Hudson et al (1984) Embo. J. 3, 2333-2339).

The peptide encoded by the H2 gene is the major stored and circulatingform in the human (Winslow et al (1992) Endrocrinology 130, 2660-2668).Hi relaxin expression is restricted to the decidua, placenta andprostate (Hansell et al (1991) J. Clin. Endocrinol. Metab. 72, 899-904),however, the H1 peptide has similar biological activity to that of H2relaxin in a rat atrial bioassay (Tan et al (1998) Br. J. Pharmacol.123, 762-770).

The actions of relaxin include an ability to inhibit myometrialcontractions, to stimulate remodelling of connective tissue and toinduce softening of the tissues of the birth canal. Additionally,relaxin increases growth and differentiation of the mammary gland andnipple and induces the breakdown of collagen, one of the main componentsof connective tissue. Relaxin decreases collagen synthesis and increasesthe release of collagenases (Unemori et al (1990) J. Biol. Chem. 265,10682-10685). These findings were recently confirmed by theestablishment of the relaxin gene-knockout mouse (Zhao et al (1999)Endocrinology 140, 445-453), which exhibited a number of phenotypicproperties associated with pregnancy. Female mice lacking a functionallyactive relaxin gene failed to relax and elongate the interpubic ligamentof the pubic symphysis and could not suckle their pups, which in turn,died within 24 hours unless cross-fostered to relaxin wildtype orrelaxin heterozygous foster mothers.

Evidence has accumulated to suggest that relaxin is more than a hormoneof pregnancy and acts on cells and tissues other than those of thefemale reproductive system. Relaxin causes a widening of blood vessels(vasodilatation) in the kidney, mesocaecum, lung and peripheralvasculature, which leads to increased blood flow or perfusion rates inthese tissues (Bani et al (1997) Gen. Pharmacol. 28, 13-22). It alsostimulates an increase in heart rate and coronary blood flow, andincreases both glomerular filtration rate and renal plasma flow (Bani etal (1997) Gen. Pharmacol. 28, 13-22). The brain is another target tissuefor relaxin where the peptide has been shown to bind to receptors(Osheroff et al (1991) Proc. Nal. Acad. Sci. U.S.A. 88, 6413-6417; Tanet al (1999) Br. J. Pharmacol 127, 91-98) in the circumventricularorgans to affect blood pressure and drinking (Parry et al (1990) JNeuroendocrinol. 2, 53-58; Summerlee et al (1998) Endocrinology 139,2322-2328; Sinnahay et al (1999) Endocrinology 140, 5082-5086).

Important clinical uses arise for relaxin in various diseases respondingto vasodilation, such as coronary artery disease, peripheral vasculardisease, kidney disease associated with arteriosclerosis or othernarrowing of kidney capillaries, or other capillaries narrowing in thebody, such as in the eyes or in the peripheral digits, the mesocaecum,lung and peripheral vasculature.

The finding of two human relaxin genes, and encoded human relaxinpeptide products nearly 20 years ago was of itself most surprising.

Even more surprisingly with the benefit of nearly 20 years of furtherresearch and development in relaxin biology internationally, theapplicant has identified, isolated and characterised nucleic acidsequences encoding a third human relaxin gene (H3), the encoded H3relaxin peptide and the constituent peptide chains thereof. Theproduction of H3 relaxin and analogues thereof has been made possible,as have uses and therapeutic treatment methods.

SUMMARY OF THE INVENTION

In a first aspect the invention relates to the peptides human H3relaxin, H3 prorelaxin and H3 preprorelaxin, to the individual peptidechains which comprise these sequences and to analogues thereof,particularly truncated and/or amino acid substituted modifications.Preferably the peptides are provided as pharmaceutically acceptablecompositions for human or animal administration, by various therapeuticroutes. Peptides are preferably isolated in purified or homogenous formfree of contaminating peptides and proteins, or in a form of about90-99% purity.

In a second aspect of the invention there is provided a compositioncomprising human H3 relaxin or a human H3 relaxin analogue having an Achain and a B chain,

the A chain having the amino acid sequence: Asp Val Leu Ala Gly Leu SerSer Ser Cys Cys Lys Trp Gly Cys Ser (SEQ ID NO: 4)1               5                   10                  15 Lys Ser GluIle Ser Ser Leu Cys             20

-   -   or an amino acid sequence truncated by up to about 9 amino acids        from N-terminus,

the B chain having the amino sequence: Arg Ala Ala Pro Tyr Gly Val ArgLeu Cys Gly Arg Glu Phe Ile Arg (SEQ ID NO: 2)1               5                   10                  15 Ala Val IlePhe Thr Cys Gly Gly Ser Arg Trp             20                  25

-   -   or an amino acid sequence truncated by up to 9 amino acids from        the amino-terminus and/or up to about 5 amino acids from the        carboxyl-terminus,    -   the A and B chains being linked by interchain disulphide bonds        at A11-B10, and A24-B22, and wherein the human H3 relaxin or        analogue thereof has relaxin bioactivity.

In a third aspect of the invention there is provided a compositioncomprising a human H3 relaxin analogue having a modified A chain and/ora modified B chain,

the H3 relaxin A chain having the amino acid sequence: Asp Val Leu AlaGly Leu Ser Ser Ser Cys Cys Lys Trp Gly Cys Ser (SEQ ID NO: 4)1               5                   10                  15 Lys Ser GluIle Ser Ser Leu Cys             20

-   -   wherein the carboxyl-terminus is an amide derivative and/or Lys        at position 12 is replaced with Glu, and/or Glu at position 19        is replaced with Gln,

the H3 relaxin B chain having the amino acid sequence: Arg Ala Ala ProTyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg (SEQ ID NO: 2)1               5                   10                  15 Ala Val IlePhe Thr Cys Gly Gly Ser Arg Trp             20                  25

-   -   wherein the carboxyl-terminus is an amide derivative,.and/ or        Ala at position 2 is replaced with Pro, and/or Arg at position 8        is replaced with Lys,    -   the A and B chains being linked by disulphide bonds between        A11-B10 and A24-B22 and wherein the human H3 relaxin analogue        has relaxin bioactivity.

In accordance with a fourth aspect of the invention there is provided acomposition comprising human H3 preprorelaxin or human H3 prorelaxin,having a signal, A chain, B chain and C chain in respect of human H3preprorelaxin, and an A chain, B chain and C chain in respect of humanH3 prorelaxin, the said amino acid chains having the amino acidsequences:

the A chain comprising: Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys LysTrp Gly Cys Ser (SEQ ID NO: 4)1               5                   10                  15 Lys Ser GluIle Ser Ser Leu Cys             20

the B chain comprising: Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly ArgGlu Phe Ile Arg (SEQ ID NO: 2)1               5                   10                  15 Ala Val IlePhe Thr Cys Gly Gly Ser Arg Trp             20                  25

the signal sequence comprising: Met Ala Arg Tyr Met Leu Leu Leu Leu LeuAla Val Trp Val Leu Thr (SEQ ID NO: 1)1               5                   10                  15 Gly Glu LeuTrp Pro Gly Ala Glu Ala             20                  25

and the C chain comprising: Arg Arg Ser Asp Ile Leu Ala His Glu Ala MetGly Asp Thr Phe Pro (SEQ ID NO: 3)1               5                   10                  15 Asp Ala AspAla Asp Glu Asp Ser Leu Ala Gly Glu Leu Asp Glu Ala            20                  25                  30 Met Gly Ser SerGlu Trp Leu Ala Leu Thr Lys Ser Pro Gln Ala Phe        35                  40                  45 Tyr Arg Gly Arg ProSer Trp Gln Gly Thr Pro Gly Val Leu Arg Gly    50                  55                  60 Ser Arg 65

In accordance with a fifth aspect of the invention there is provided acomposition comprising the C chain of human H3 relaxin, the C chainhaving the amino acid sequence: Arg Arg Ser Asp Ile Leu Ala His Glu AlaMet Gly Asp Thr Phe Pro (SEQ ID NO: 3)1               5                   10                  15 Asp Ala AspAla Asp Glu Asp Ser Leu Ala Gly Glu Leu Asp Glu Ala            20                  25                  30 Met Gly Ser SerGlu Trp Leu Ala Leu Thr Lys Ser Pro Gln Ala Phe        35                  40                  45 Tyr Arg Gly Arg ProSer Trp Gln Gly Thr Pro Gly Val Leu Arg Gly    50                  55                  60 Ser Arg 65

In accordance with a sixth aspect of the invention there is provided anucleic acid sequence encoding human prepro-H3 relaxin comprising thenucleic acid sequence: tataaatggg gggccaagag gcagcagaga cactggcccactctcacgtt caaagcgtct 60 (SEQ ID NO: 6) ccgtccagca tggccaggta catgctgctgctgctcctgg cggtatgggt gctgaccggg 120 gagctgtggc cgggagctga ggcccgggcagcgccttacg gggtcaggct ttgcggccga 180 gaattcatcc gagcagtcat cttcacctgcgggggctccc ggtggagacg atcagacatc 240 ctggcccacg aggctatggg tgaggctggggagagagtgg atgtagaagg ggaacaggtg 300 gctggatggg tcccaggagc taaggacagagataagagga ggttgctgga ggaggagggt 360 ccctgtcctg ccacattcag ccagggacacctgcccagcc ttgaaacaag ggctcaggag 420 ttagcagagc tgcagagctg ggatggggtgttgcaagcca tccatggggg ctggaagtct 480 gaggacaggt gggggcgggg agcgtgccatttgcaaagac aacaccgaag tgttttccaa 540 ccctttccag caggtaatgt gaagggtgtggtatacacat agctgggttt gtcacctaat 600 gcatgacctc tccccagcaa gttggtttttcttccgtctc tgagtgtctt ttttttggag 660 atgtggtctc actccattgc ccaggcttgaatgcagtggc ccaatcactg ctcattgcag 720 cctcgacctc ccaggctcaa gtgattctcctgcctccgcc tccagagtag ttgagaccac 780 aggcacctga caccatgcct ggctagttttaaattttttt tttgtagaaa caggggtctc 840 actatgttgc ctaggctggt ctcgaactcctgggctcaag tgatcctccc acctcggcct 900 ccctaagtgc tgagattaga gtctctgagtgtctttatct tcaaatggga gacacagttc 960 ctgaatcttg caggattaag tggtatgattaaatcaaaac agattagggc agagtctcag 1020 cagggcagcg gcacaatctg ggatccatcaggagagtcag agggaacaga agacctagct 1080 tcatgagggg cagggacctg gcaaatagatattcatgatg gtgagaagga ggataggtat 1140 gagcgtggac atagaagaca caccacttggattcagatag tagctctaca atgtaatagt 1200 tgtgtgttca tgtgctacta ttttttttttttttgagaca gaatctcatt ctgttgccca 1260 ggctggagtg cagtggtgca atcttggctcactgtaacct ccatcacctg ggttcaagcg 1320 attctcgtgc ctccagcctc ccaagtagctgggattacag atgtgtgcca ccatacctcg 1380 ctaatctttt tatttttagt agagacagtttcaccatgtt ggccaggctg gtctccaact 1440 cctgacctca ggtgatcctc ccacctcagcctcccaaagt gctgggatta caggcatgag 1500 ccaccgcgcc cagccatgca aattctttactgagtcctgc ctcagtggtc tcctctggaa 1560 aatacgggtg ataactgcac ccacctcaactggttatcac tgagaagaat aaagaagtta 1620 acctgctaaa gcacttaaaa cgttgtttgacacaaagtaa gtgatcaata aattattatt 1680 attattatta ttattattat tattattatttttgagacag ggtcttgctc tgttgcccag 1740 actggagtgc agtggtgtga tcacagctcactgcagcttc aacctcttgg gctcaagcaa 1800 ttctcctgcc tcagcctcct gagtagctgggactacaggc ttgtgccaac atgtctaact 1860 ttttattatt tgtagagaca gggtagtgctgtgttgtcca ggctgttctt gaactcctgg 1920 ttctggtgat cctccagcat gtgcccctggaagtgctggg attacaggtg tgagacaccg 1980 tgcccggact caatagtcat ttttgagtgctcatcatgtt ccagacattg ttctaagttt 2040 ttttttttaa tgaatattaa ctccttataaaacttgagaa ggttggagta attatttttt 2100 tccactttgc agaaaagaac attgaggctccaagaagtaa atttacttgc tcacgattag 2160 agaagctgga ttcatgctca gtcagcccagctcccaaatg taccaggtcc tcaattaata 2220 aagagtaagg agaaataaat gacagggctgggtgcggtgg ctcacgcctg taatcccagc 2280 actttgggtg gctgaggtgg gcacatcacttgaggtcagg agtttgcgac cagcctgaac 2340 aacatggtga accccatctc tataacaatacaaaaatcag ccaggcctgc tggcagacac 2400 ctgtaatccc acctactctg gcagagccagaatttgaacc caggactggg tggaataaaa 2460 actctgaact atgtctatga ctgttgtcacaagatcagag ctagactggc caggagccat 2520 gactgtgggt gcagcagcag ctgagccctgatcactaact ctgttcatct tttgcaggag 2580 ataccttccc ggatgcagat gctgatgaagacagtctggc aggcgagctg gatgaggcca 2640 tggggtccag cgagtggctg gccctgaccaagtcacccca ggccttttac agggggcgac 2700 ccagctggca aggaacccct ggggttcttcggggcagccg agatgtcctg gctggccttt 2760 ccagcagctg ctgcaagtgg gggtgtagcaaaagtgaaat cagtagcctt tgctagtttg 2820 agggctgggc agccgtgggc accaggaccaatgccccagt cctgccatcc actcaactag 2880 tgtctggctg ggcacctgtc tttcgagcctcacacattca ttcattcatc tacaagtcac 2940 agaggcactg tgggctcagg cacagtctcccgacaccacc tatccaaccc tgccctttga 3000 ccagcctatc atgaccctgg cccctaaggaagctgtgccc ctgcctggtc aagtggggac 3060 ccccccatcc tgacccctga cctctccccagccctaacca tgcgtttgcc tggcctacac 3120 actccactgc cacaactggg tccctactctacctaggctg gccacacaga gacccctgcc 3180 cccttcccag tccaaactgt ggccattgtcccctgaccag ctaaaatcaa gcctctgtct 3240 cagtccagcc tttgcacgca cgcttcctttgccctgcttt ccatcccctc tccctccaac 3300 tcccctgcca gagttccaag gctgtggaccccagagaagg tggcaggtgg cccccctagg 3360 agagctctgg gcacattcga atcttcccaaactccaataa taaaaattcg aagactttgg 3420 cagagagtgt gtgtgtgtgt gtatggttg3449

In accordance with a seventh aspect of the invention there is provided anucleic acid sequence encoding human pro-H3 relaxin including an Achain, B chain and C chain sequence,

the A chain sequence comprising: gatgtcctgg ctggcctttc cagcagctgc 60(SEQ ID NO: 7) tgcaagtggg ggtgtagcaa aagtgaaatc agtagccttt gc 72

the B chain sequence comprising: cgggcagcgc cttacggggt caggctttgc 60(SEQ ID NO: 8) ggccgagaat tcatccgagc agtcatcttc acctgcgggg gctcccggtg g81

the C chain sequence comprising: agacgatcag acatcctggc ccacgaggct 60(SEQ ID NO: 9) atgggagata ccttcccgga tgcagatgct gatgaagaca gtctggcaggcgagctggat 120 gaggccatgg ggtccagcga gtggctggcc ctgaccaagt caccccaggccttttacagg 180 gggcgaccca gctggcaagg aacccctggg gttcttcggg gcagccga 198

In an eighth aspect of the invention there is provided a nucleic acidsequence encoding human H3 relaxin having an A and B chain,

the A chain sequence comprising: gatgtcctgg ctggcctttc cagcagctgc 60(SEQ ID NO: 7) tgcaagtggg ggtgtagcaa aagtgaaatc agtagccttt gc 72

and the B chain sequence comprising: cgggcagcgc cttacggggt caggctttgc 60(SEQ ID NO: 8) ggccgagaat tcatccgagc agtcatcttc acctgcgggg gctcccggtg g81

In a ninth aspect of the invention there is provided a nucleic acidsequence encoding the A, B or C peptide chains of human H3 relaxin, thesaid chains comprising the nucleic acid sequences:

A chain: gatgtcctgg ctggcctttc cagcagctgc 60 (SEQ ID NO: 7) tgcaagtgggggtgtagcaa aagtgaaatc agtagccttt gc 72

B chain: cgggcagcgc cttacggggt caggctttgc 60 (SEQ ID NO: 8) ggccgagaattcatccgagc agtcatcttc acctgcgggg gctcccggtg g 81

and C chain: agacgatcag acatcctggc ccacgaggct 60 (SEQ ID NO: 9)atgggagata ccttcccgga tgcagatgct gatgaagaca gtctggcagg cgagctggat 120gaggccatgg ggtccagcga gtggctggcc ctgaccaagt caccccaggc cttttacagg 180gggcgaccca gctggcaagg aacccctggg gttcttcggg gcagccga 198

The nucleic acid sequences are isolated and purified nucleic acids, andmay be contained within a vector, such as a plasmid, bacteriophage orvirus DNA or RNA, and may be in single or double stranded form, and mayinclude promoters or enhancers or other sequences which confer elevated,enhanced or other effects on expression in a host system such as abacterial cell.

The triplet codons of nucleic acids encode specific amino acids. Morethan one codon may encode the same amino acid, as is well andestablished in the art. Moreover, methods of modifying or altering thesequence of nucleic acids are well known in the art. Insofar as thisinvention pertains in its various aspects to nucleic acids encodinghuman H3 relaxin, pro-H3 relaxin, prepro-H3 relaxin, and constituentpeptide chains thereof, the invention includes nucleic acid variantswhich encode the same protein products, or a protein product havingrelaxin activity.

Nucleotide sequence aspects of this invention also include closelyrelated nucleic acid sequences as defined by stringent hybridization,this being annealing of complimentary sequences under conditions of0.25M H₂PO₄, pH 7.2, 1 mmol EDTA, 20% SDS at 65° C. overnight; followedby 3 washes for 5 min in 2×SSC, 0.1% SDS at room temperature; andfinally a 30 min wash at 65° C. in 0.1% SSC; where 6×SSC is 0.9M NaCl,0.3M Na₃CO₂ H₂O at ph 7.0. Such sequences will encode H3 relaxinpolypeptides having biological or immunological or other activitycorresponding to those of H3 relaxin.

In another aspect of the invention there is provided a method for thetreatment of one or more of: vascular disease including coronary arterydisease, peripheral vascular disease, vasospasm including Raynaud'sphenomenon, microvascular disease involving the central and peripheralnervous system, kidney, eye and other organs; treatment of arterialhypertension; diseases related to uncontrolled or abnormal collagen orfibronectin formation such as fibrotic disorders (including fibrosis oflung, heart and cardiovascular system, kidney and genitourinary tract,gastrointestinal system, cutaneous, rheumatologic and hepatobiliarysystems); kidney disease associated with vascular disease, interstitialfibrosis, glomerulosclerosis, or other kidney disorders; psychiatricdisorders including anxiety states including panic attack, agoraphobia,global anxiety, phobic states; depression or depressive disordersincluding major depression, dysthymia, bipolar and unipolar depression;neurologic or neurodegenerative diseases (including memory loss or othermemory disorders, dementias, Alzheimer's disease); disorders oflearning, attention and motivation (including Attention DeficitHyperactivity Disorder, Tourette's disease, impulsivity, antisocial andpersonality disorders, negative symptoms of psychoses including thosedue to schizophrenia, acquired brain damage and frontal lobe lesions);addictive disorders (including drug, alcohol and nicotine addiction);movement and locomotor disorders (including Parkinson's disease,Huntington's disease, and motor deficits after stoke, head injury,surgery, tumour or spinal cord injury); immunological disorders(including immune deficiency states, haematological andreticuloendothelial malignancy, breast disorders (including fibrocysticdisease, impaired lactation, and cancer); endometrial disordersincluding infertility due to impaired implantation; endocrine disorders(including adrenal, ovarian and testicular disorders related to steroidor peptide hormone production) ; delayed onset of labour, impairedcervical ripening, and prevention of prolonged labour due to fetaldystocia; sinus bradycardia; hair loss, alopecia; disorders of waterbalance including impaired or inappropriate secretion of vasopressin;placental insufficiency; which comprises administering to a subject inneed of any such treatments a therapeutically effective amount of humanH3 relaxin, or an analogue thereof as herein defined, optionally inassociation with one or more pharmaceutically acceptable carriers and/diluents and/or excipients.

In another aspect of the invention there is provided use of human H3relaxin or an analogue thereof in the manufacture of medicaments for thetreatment of one or more of: vascular disease including coronary arterydisease, peripheral vascular disease, vasospasm including Raynaud'sphenomenon, microvascular disease involving the central and peripheralnervous system, kidney, eye and other organs; treatment of arterialhypertension; diseases related to uncontrolled or abnormal collagen orfibronectin formation such as fibrotic disorders (including fibrosis oflung, heart and cardiovascular system, kidney-and genitourinary tract,gastrointestinal system, cutaneous, rheumatologic and hepatobiliarysystems); kidney disease associated with vascular disease, interstitialfibrosis, glomerulosclerosis, or other kidney disorders; psychiatricdisorders including anxiety states including panic attack, agoraphobia,global anxiety, phobic states; depression or depressive disordersincluding major depression, dysthymia, bipolar and unipolar depression;neurologic or neurodegenerative diseases (including memory loss or othermemory disorders, dementias, Alzheimer's disease); disorders oflearning, attention and motivation (including Attention DeficitHyperactivity Disorder, Tourette's disease, impulsivity, antisocial andpersonality disorders, negative symptoms of psychoses including thosedue to schizophrenia, acquired brain damage and frontal lobe lesions);addictive disorders (including drug, alcohol and nicotine addiction);movement and locomotor disorders (including Parkinson's disease,Huntington's disease, and motor deficits after stoke, head injury,surgery, tumour or spinal cord injury); immunologicaldisorders(including immune deficiency states, haematological andreticuloendothelial malignancy, breast disorders (including fibrocysticdisease, impaired lactation, and cancer); endometrial disordersincluding infertility due to impaired implantation; endocrine disorders(including adrenal, ovarian and testicular disorders related to steroidor peptide hormone production); delayed onset of labour, impairedcervical ripening, and prevention of prolonged labour due to fetaldystocia; sinus bradycardia; hair loss, alopecia; disorders of waterbalance including impaired or inappropriate secretion of vasopressin;placental insufficiency; which comprises administering to a subject inneed of any such treatments a therapeutically effective amount of humanH3 relaxin, or an analogue thereof as herein defined, optionally inassociation with one or more pharmaceutically acceptable carriers and/diluents and/or excipients. Sequence Listing Table SEQ ID NO: 1 Signalpeptide sequence SEQ ID NO: 2 B chain peptide sequence SEQ ID NO: 3 Cchain peptide sequence SEQ ID NO: 4 A chain peptide sequence SEQ ID NO:6 Genomic DNA sequence SEQ ID NO: 7 A chain nucleic acid sequence SEQ IDNO: 8 B chain nucleic acid sequence SEQ ID NO: 9 C chain nucleic acidsequence

DESCRIPTION OF THE FIGURES

FIG. 1. Assembled DNA sequence of the H3 (A) and M3 (B) genes.

Start and Stop codons as well as predicted TATA boxes andpolyadenylation sequences are underlined. The positions of the putativesignal peptide, and B-, C- and A-chain peptide sequences are indicatedby arrows. A- and B-chain sequences are boxed and the residuesimplicated in relaxin receptor binding are shaded. The intron sequence,which is at an identical position in the C-chain in both the human (A)and mouse (B) sequences, is indicated by lower case letters and theexact size of the intron is marked.

FIG. 2. Sequence comparisons of H3 and M3 relaxin with other relaxin andinsulin family members.

Alignments of A- and B-chain sequences from H3 and M3 relaxin with otherhuman and mouse relaxin sequences (A). The consensus sequences areboxed; Cons 1,2,3: Consensus sequence between human relaxins 1, 2 and 3.Cons 3: Consensus sequence between H3 and M3 relaxin for the B-chain andH3, R3 and M3 relaxin for the A-chain. Cons Mouse: Consensus sequencebetween M1 and M3 relaxin. The rat sequence is derived from an EST clone(see results for details). “+” Denotes a conservative substitution, “.”denotes no homology. Phylogenetic tree of evolution of H3 and M3 relaxinfull-length sequences with human sequences of the relaxin/insulin/IGFsuperfamily (B).

FIG. 3. Bioactivity of H3 compared to H1 and H2 relaxin in a humanrelaxin receptor expressing cell line.

cAMP accumulation in THP-1 cells upon stimulation with peptides (A).Data are expressed as mean ±SEM of the maximum response (%) to H2relaxin (n=3). The response to bovine insulin (bINSL) and human INSL3(hINSL3) are also shown to highlight the specificity of the assay. H1,H2, H3; Human 1, 2 and 3 relaxin respectively. The ability of H1 (n=7),H2 (n=11) and H3 (n=3) relaxin peptides to compete for ³³P-labeled H2relaxin (B33) binding to THP-1 cells (B). Data are expressed as mean±SEMof the specific binding (%).

FIG. 4. Ability of a well characterized H2 relaxin antibody to recognizeH3 relaxin.

The H2 relaxin antibody was immobilized onto ELISA plates and acompetition experiment was performed using H1, H2 and H3 relaxin against¹²⁵I-labeled H2 relaxin. Results are mean±SEM of the specific binding(%) of triplicate determinations from a representative assay.

Unexpectedly, some 20 years after the identification of human relaxin,and the surprising identification at that time of two human relaxingenes, a further relaxin gene has been identified. This invention in itsvarious aspects provides: the characterisation of nucleotide sequencesencoding human H3 relaxin; the isolation of purified nucleic acidmaterial; amplification of nucleotide sequences encoding H3 relaxin(mRNA, cDNA and DNA); nucleic acid cloning of H3 relaxin sequences;nucleic acid sequence identification, and peptide sequences encodinghuman H3 preprorelaxin, H3 prorelaxin and H3 relaxin.

The human H3 relaxin polypeptide comprises disulphide bridged A and Bchains. The amino acid sequence of human H3 relaxin is set out in SEQ IDNO: 4. The amino acid sequence of the B chain of human relaxin is setout in SEQ ID NO: 2.

The A and B chains of human H3 relaxin are linked through cysteineresidues, A11-B10, A24-B22 disulphide bond formation taking placebetween these cysteine linkages.

Hence, the amino acid sequence of human H3 relaxin A and B chains are asfollows: A Chain (SEQ ID NO: 4) Asp Val Leu Ala Gly Leu Ser Ser1               5 Ser Cys Cys Lys Trp Gly Cys Ser    10                  15 Lys Ser Glu Ile Ser Ser Leu Cys            20

B Chain (SEQ ID NO: 2) Arg Ala Ala Pro Tyr Gly Val Arg Leu Cys Gly1               5                   10 Arg Glu Phe Ile Arg            15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg Trp            20                  25

-   -   the A and B chains being linked by disulphide bonds between        A11-B10, A24-B22.

Human H3 relaxin possesses classical relaxin bioactivity. Humanrelaxins, H1 and H2 relaxin, bind to cells expressing relaxin receptors,such as THP-1 cells (Parsell et al (1996) J. Biol. Chem. 271,27936-27941). H2 relaxin produces a dose dependent increase in cAMPproduction from these cells. Synthetic H3 relaxin produced according tothis invention stimulated a dose dependent increase in cAMP in keepingwith human H2 relaxin. The specificity of response in target cellsbearing the human relaxin receptor as exhibited by H3 relaxin isdemonstrated by the inability of bovine insulin (bINSL) or human insulin(hINSL3) to stimulate cAMP responses at doses up to 1 um in THP-1 cells.

The elicitation of a second messager response (cAMP) by stimulatinghuman relaxin receptors with human H3 relaxin, provides definitiveevidence that human H3 relaxin has classic relaxin biological activity.Such assays in cells containing relaxin receptors, for example THP-1cells as referred to above provides, a ready way to determine relaxinactivity. In addition, the ability of human H3 relaxin to compete withP³²-labelled H2 relaxin in binding to relaxin binding sites in cellsexpressing relaxin receptors, again provides definitive confirmation ofrelaxin activity.

Other biological activities/assays for determining relaxin activity areknown in the art. For example, bioassays used for the measurement ofactive relaxin during pregnancy and non-pregnancy, such as the guineapig interpubic ligament assay may be used (Steinetz et al (1960)Endocrinology 67, 102-115, and Sirosi et al (1983) American Journal ofObstetrics and Gynaecology 145: 402405) may be used. Other bioassaysinclude cAMP production in THP-1 cells (Parsell et al (1996) J. Biol.Chem 271, 27936-27941).

Applicant has found that H3 relaxin analogues may be prepared where upto 9 amino acids are truncated from the N-terminus of the A chain, andup to 9 amino acids are truncated from the N-terminus of the B chain,and up to 5 amino acids are truncated from the C-terminus of the Bchain.

The resulting relaxin analogues comprise a H3 relaxin A and B chain, theA chain having the amno acid sequence Asp Val Leu Ala Gly Leu Ser Ser(SEQ ID NO: 4) 1               5 Ser Cys Cys Lys Trp Gly Cys Ser    10                  15 Lys Ser Glu Ile Ser Ser Leu Cys            20

-   -   truncated by up to about 9 amino acids from amino-terminus,

and the B chain having the amino acid sequence: (SEQ ID NO: 2) Arg AlaAla Pro Tyr Gly Val Arg Leu Cys Gly1               5                   10 Arg Glu Phe Ile Arg            15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg Trp            20                  25

-   -   truncated by up to 9 amino acids from the amino-terminus and/or        up to about 5 amino acids from the carboxyl-terminus,    -   the A and B chains being linked by disulphide bonds between        A11-B10 and A24-B22, and wherein the human H3 relaxin or        analogue thereof has relaxin bioactivity. The A chain of human        H3 relaxin contains an intrachain disulphide bond between Cys        residues 10 and 15.

In standard assays looking at second messenger elicitation in cellsbearing human relaxin receptors, the H3 relaxin analogues referred toabove all elicited cyclic AMP production in a manner which wascharacteristic of full length, non-truncated human H3 relaxin, andindeed human H2 relaxin. Hence, such truncated H3 relaxin analoguespossess relaxin bioactivity.

Another aspect of the present invention relates to compositionscomprising a human H3 relaxin analogue having a modified A chain and/ora modified B chain. The carboxyl-terminus of the A chain, and/or the Bchain, may be an amide derivative. Lys at position 12 in the A chain maybe replaced with Glu, and/or Glu at position 19 may be replaced withGln. In the B chain, the Ala at position 2 may be replaced with Pro,and/or Arg at position 8 may be replaced with Lys. The resulting H3relaxin analogues having modified amino acids comprise an amino acidsequence which may be depicted as follows:

In accordance with another aspect of the invention, there is provided ahuman H3 relaxin analogue having a modified A chain and/or a modified Bchain,

the H3 relaxin A chain having the amino acid sequence: Asp Val Leu AlaGly Leu Ser Ser Ser (SEQ ID NO: 4) 1               5 Cys Cys Lys Trp GlyCys Ser 10                  15 Lys Ser Glu Ile Ser Ser Leu Cys            20

-   -   wherein the carboxyl-terminus is an amide derivative and/or Lys        at position 12 is replaced with Glu, and/or Glu at position 19        is replaced with Gln,

the modified B chain having the amino acid sequence: (SEQ ID NO: 2) ArgAla Ala Pro Tyr Gly Val Arg Leu Cys Gly1               5                   10 Arg Glu Phe Ile Arg            15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg Trp            20                  25

-   -   wherein the carboxyl-terminus is an amide derivative, and/or Ala        at position 2 is replaced with Pro, and/or Arg at position 8 is        replaced with Lys,    -   the A and B chains being linked by disulphide bonds between        A11-B10 and A24-B22, and wherein the human H3 relaxin analogue        has relaxin bioactivity.

The isolation, purification and characterisation of nucleic acidsequences encoding human H3 relaxin has allowed the characterisation andproduction of the signal sequence of human H3 relaxin, and thepro-sequence of human H3 relaxin.

The identification, purification and characterisation of the signalsequence and C chain of human H3 relaxin enables the prepro- andpro-human H3 relaxin to be produced.

In accordance with another aspect of the invention there is provided acomposition comprising human H3 preprorelaxin or human H3 prorelaxin,having a signal, A chain, B chain and C chain in respect of human H3preprorelaxin, and an A chain, B chain and C chain in respect of humanH3 prorelaxin, the said amino acid chains having the amino acidsequences:

the A chain comprising: Asp Val Leu Ala Gly Leu Ser Ser (SEQ ID NO: 4)1               5 Ser Cys Cys Lys Trp Gly Cys Ser    10                  15 Lys Ser Glu Ile Ser Ser Leu Cys            20

the B chain comprising: (SEQ ID NO: 2) Arg Ala Ala Pro Tyr Gly Val ArgLeu Cys Gly 1               5                   10 Arg Glu Phe Ile Arg            15 Ala Val Ile Phe Thr Cys Gly Gly Ser Arg Trp            20                  25

the signal sequence comprising: Met Ala Arg Tyr Met Leu Leu Leu Leu (SEQID NO: 1) 1               5 Leu Ala Val Trp Val Leu Thr10                  15 Gly Glu Leu Trp Pro Gly Ala Glu Ala            20                  25

and the C chain comprising: Arg Arg Ser Asp Ile Leu Ala His Glu Ala MetGly Asp Thr Phe Pro (SEQ ID NO: 3)1               5                   10                  15 Asp Ala AspAla Asp Glu Asp Ser Leu Ala Gly Glu Leu Asp Glu Ala            20                  25                  30 Met Gly Ser SerGlu Trp Leu Ala Leu Thr Lys Ser Pro Gln Ala Phe        35                  40                  45 Tyr Arg Gly Arg ProSer Trp Gln Gly Thr Pro Gly Val Leu Arg Gly    50                  55                  60 Ser Arg 65

In accordance with a further aspect of the invention there is providedthe C chain of human H3 relaxin, said C chain having the amino acidsequence: Arg Arg Ser Asp Ile Leu Ala His Glu Ala Met Gly Asp Thr PhePro (SEQ ID NO: 3)1               5                   10                  15 Asp Ala AspAla Asp Glu Asp Ser Leu Ala Gly Glu Leu Asp Glu Ala            20                  25                  30 Met Gly Ser SerGlu Trp Leu Ala Leu Thr Lys Ser Pro Gln Ala Phe        35                  40                  45 Tyr Arg Gly Arg ProSer Trp Gln Gly Thr Pro Gly Val Leu Arg Gly    50                  55                  60 Ser Arg 65

Human H3 prorelaxin possesses characteristic relaxin bioactivity.

Human H3 relaxin, prorelaxin, preprorelaxin and constitutive peptidechains may be products using techniques previously described as usefulin the production of relaxin such as U.S. Pat. No. 5,991,997, U.S. Pat.No. 4,758,516, U.S. Pat. No. 4,871,670, U.S. Pat. No. 4,835,251,PCT/US90/02085, and PCT/US94/0699.

Relaxin analogues and derivatives where amino acids are substituted asindicated above may be produced recombinantly using, for example, sitedirected mutagenesis techniques as set forth, for example, in Tsurushitaet al (1988) Gene Tissue: 135-139.

The disclosed sequence information for human H3 relaxin, analoguesthereof wherein one or more amino acids are truncated from the N- and/orC-terminus of the A and/or B chains, or human H3 relaxin analogueshaving amino acid substitutions as referred to above, may be synthesisedaccording to the methods of Büllesbach (1991) J. Biol. Chem. 266,10754-10761, for synthesising relaxin. Additionally, well known methodsof peptide synthesis may be utilised to produce the various H3 relaxinforms referred to herein.

Relaxin has been implicated consequently in the treatment and diagnosisof various diseases and disorders. For example, studies provide evidencethat relaxin is effective in the treatment of scleroderma, sinusbradycardia, cardiovascular disease, neurodegenerative and neurologicdisorders, hair loss, depression. See e.g., U.S. Pat. No. 5,166,191 andInternational Patent Application No. PCT/US92/069). Evidence alsosuggests the use of relaxin in diseases and disorders related to theabnormal expression of collagen or fibronectin, such as rheumatoidarthritis.

Human H3 relaxin, human H3 relaxin truncated analogues, amino acidmodified H3 relaxin analogues, and human prorelaxin provided by theinstant invention bind to the relaxin receptor and possess relaxinbiological activity. It directly follows that these human H3 relaxinforms possessing relaxin biological activity may be used for thetreatment of the above-identified diseases and other diseases.

In accordance with another aspect of the present invention there isprovided a method for the treatment of one or more of: vascular diseaseincluding coronary artery disease, peripheral vascular disease,vasospasm including Raynaud's phenomenon, microvascular diseaseinvolving the central and peripheral nervous system, kidney, eye andother organs; treatment of arterial hypertension; diseases related touncontrolled or abnormal collagen or fibronectin formation such asfibrotic disorders -(including fibrosis of lung, heart andcardiovascular system, kidney and genitourinary tract, gastrointestinalsystem, cutaneous, rheumatologic and hepatobiliary systems); kidneydisease associated with vascular disease, interstitial fibrosis,glomerulosclerosis, or other kidney disorders; psychiatric disordersincluding anxiety states including panic attack, agoraphobia, globalanxiety, phobic states; depression or depressive disorders includingmajor depression, dysthymia, bipolar and unipolar depression; neurologicor neurodegenerative diseases (including memory loss or other memorydisorders, dementias, Alzheimer's disease); disorders of learning,attention and motivation (including Attention Deficit HyperactivityDisorder, Tourette's disease, impulsivity, antisocial and personalitydisorders, negative symptoms of psychoses including those due toschizophrenia, acquired brain damage and frontal lobe lesions);addictive disorders (including drug, alcohol and nicotine addiction);movement and locomotor disorders (including Parkinson's disease,Huntington's disease, and motor deficits after stoke, head injury,surgery, tumour or spinal cord injury); immunological disorders(including immune deficiency states, haematological andreticuloendothelial malignancy; breast disorders (including fibrocysticdisease, impaired lactation, and cancer); endometrial disordersincluding infertility due to impaired implantation; endocrine disorders(including adrenal, ovarian and testicular disorders related to steroidor peptide hormone production) ; delayed onset of labour, impairedcervical ripening, and prevention of prolonged labour due to fetaldystocia; sinus bradycardia; hair loss, alopecia; disorders of waterbalance including impaired or inappropriate secretion of vasopressin;placental insufficiency; which comprises administering to a subject inneed of any such treatments a therapeutically effective amount of humanH3 relaxin, or an analogue thereof as herein defined, optionally inassociation with one or more pharmaceutically acceptable carriers and/diluents and/or excipients.

In accordance with another aspect of the present invention there isprovided use of human H3 relaxin or an analogue thereof in themanufacture of medicaments for the treatment of one or more of: vasculardisease including coronary artery disease, peripheral vascular disease,vasospasm including Raynaud's phenomenon, microvascular diseaseinvolving the central and peripheral nervous system, kidney, eye andother organs; treatment of arterial hypertension; diseases related touncontrolled or abnormal collagen or fibronectin formation such asfibrotic disorders (including fibrosis of lung, heart and cardiovascularsystem, kidney and genitourinary tract, gastrointestinal system,cutaneous, rheumatologic and hepatobiliary systems); kidney diseaseassociated with vascular disease, interstitial fibrosis,glomerulosclerosis, or other kidney disorders; psychiatric disordersincluding anxiety states including panic attack, agoraphobia, globalanxiety, phobic states; depression or depressive disorders includingmajor depression, dysthyria, bipolar and unipolar depression; neurologicor neurodegenerative diseases (including memory loss or other memorydisorders, dementias, Alzheimer's disease); disorders of learning,attention and motivation (including Attention Deficit HyperactivityDisorder, Tourette's disease, impulsivity, antisocial and personalitydisorders, negative symptoms of psychoses including those due toschizophrenia, acquired brain damage and frontal lobe lesions);addictive disorders (including drug, alcohol and nicotine addiction);movement and locomotor disorders (including Parkinson's disease,Huntington's disease, and motor deficits after stoke, head injury,surgery, tumour or spinal cord injury); immunological disorders(including immune deficiency states, haematological andreticuloendothelial malignancy; breast disorders (including fibrocysticdisease, impaired lactation, and cancer); endometrial disordersincluding infertility due to impaired implantation; endocrine disorders(including adrenal, ovarian and testicular disorders related to steroidor peptide hormone production) ; delayed onset of labour, impairedcervical ripening, and prevention of prolonged labour due to fetaldystocia; sinus bradycardia; hair loss, alopecia; disorders of waterbalance including impaired or inappropriate secretion of vasopressin;placental insufficiency; which comprises administering to a subject inneed of any such treatments a therapeutically effective amount of humanH3 relaxin, or an analogue thereof as herein defined, optionally inassociation with one or more pharmaceutically acceptable carriers and/diluents and/or excipients.

Without wishing to be bound on mechanism of action, applicant believesthat H3 relaxin may act as a neurotransmitter or neuroregulator in thebrain, and other parts of the body including nerves, for example throughinducing eAMP production in cells. H3 relaxin may also allow nutrientuptake by cells, or may be involved in autoregulatory presynaptic and/orconventional postsynaptic actions. Applicant further believes that H3relaxin may also be axonally transported by nerve projections.

As defined hereinafter, H3 relaxin has surprisingly been found to beexpressed in neuroanatomical region of the pars ventromedialis of thedorsal tegmental nucleus (vmDTg), which may otherwise be referred to asthe nucleus incertus (Goto et al (2001) Journal of Comparative Neurology438: 86-122). With the extensive pattern of efferents and afferents toand from key forebrain areas from the nucleus incertus, this region hasbeen proposed as part of a brain stem network that may regulatebehavioural activation via influences on attention, motivation,locomotion and learning (Goto et al) and may give rise to thetherapeutic treatment modalities herein described. This is consistentwith the abundent distribution of relaxin binding sites in cerebralcortex and other relevant brain areas (Osheroff and Phillips (1991)Proc. Natl. Acad. Sci. USA 88, 6413-6417; and Tan et al (1999) Br. J.Pharmacol. 127, 91-98).

H3 relaxin may cross the blood brain barrier, or may be treated tofacilitate crossing of the blood brain barrier, by methods known in theart incluidng use of one or more sugars or amino acids, or othersubstances which open the blood brain barrier or make it leaky allowingcoadministered/timed administration with H3 relaxin (see for exampleNaito U.S. Pat. No. 6,294,520), by intranasal administration accordingto the methods of Frey (U.S. Pat. No. 6,313,093), for example using alipophilic vehicle, and by methods described in PCT/WO89/10134.

H3 relaxin and anlogues as herein described may be effective in thetreatment of a wide range of what may broadly be described as neurologicdiseases including psychiatric disorders, disorders of learning,attention and memory, addictive disorders and movement and locomotordisorders.

H3 relaxin binds to the relaxin receptor as described hereinafter.

For convenience, human H3 relaxin, analogues of human H3 relaxin whereone or more amino acids are truncated from the N— and/or C-terminus ofthe A and B chains of human H3 relaxin, analogues of human H3 relaxinwhere one or more amino acids are modified or substituted with anotheramino acid as described herein, and human H3 preprorelaxin shallcollectively be referred to as human H3 relaxin, unless otherwisespecifically indicated.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes the IC.sub.50as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms or a prolongation of survivalin a patient. Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD.sub.50 (the doselethal to 50% of the population) and the ED.sub.50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD.sub.50 and ED.sub.50. Compoundswhich exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED.sub.50 with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g. Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p1).

Dosage amount and interval may be adjusted individually to provide serumlevels of the active moiety which are sufficient to maintain the relaxinactivity and effects.

Administration of H3 relaxin can be via any of the accepted modes ofadministration for agents that serve similar utilities, preferably bysystemic administration.

While human dosage levels for treating many of the above-identifiedrelaxin related diseases or disorders have yet to be optimized for H3relaxin generally, a daily dose is from about 0.05 to 500.0 .mu.g/kg ofbody weight per day, preferably about 5.0 to 200.0 .mu.g/kg, and mostpreferably about 10.0 to 100.0 .mu.g/kg. Generally it is sought toobtain a serum concentration of the H3 relaxin approximating or greaterthan normal circulating levels of relaxin in pregnancy, i.e., 1.0 ng/ml,such as 1.0 to 20 ng/ml, preferably 1.0 to 20 ng/ml.

For administration to a 70 kg person, the dosage range would be about7.0 .mu.g to 3.5 mg per day, preferably about 42.0 .mu.g to 2.1 mg perday, and most preferably about 84.0 to 700.0 .mu.g per day. The amountof the H3 relaxin administered will, of course, be dependent on thesubject and the severity of the affliction, the manner and schedule ofadministration and the judgment of the prescribing physician and thebiological activity of such analog or derivative. One treatment regimencan employ a higher initial dosage level (e.g., 100 to 200 .mu.g/kg/day)followed by decreasing dosages to achieve steady H3 relaxin serumconcentration of about 1.0 ng/ml. Another treatment regimen,particularly postpartum depression, entails administration of an amountof H3 relaxin sufficient to attain normal pregnancy levels of relaxin(about 1.0 ng/ml) followed by gradual decreasing dosages until H3relaxin serum levels are no longer detectable (e.g. less than about 20picograms/ml), optionally discontinuing treatment upon reaching thatdosage level.

Any pharmaceutically acceptable mode of administration can be used. H3relaxin can be administered either alone or in combination with otherpharmaceutically acceptable excipients, including solid, semi-solid,liquid or aerosol dosage forms, such as, for example, tablets, capsules,powders, liquids, gels, suspensions, suppositories, aerosols or thelike. Such proteins can also be administered in sustained or controlledrelease dosage forms (e.g., employing a slow release bioerodabledelivery system), including depot injections, osmotic pumps (such as theAlzet implant made by Alza), pills, transdermal (includingelectrotransport) patches, and the like, for prolonged administration ata predetermined rate, preferably in unit dosage forms suitable forsingle administration of precise dosages. The compositions willtypically include a conventional pharmaceutical carrier or excipientand/or H3 relaxin, H3 prorelaxin, and H3 preprorelaxin or derivativesthereof. In addition, these compositions may include other activeagents, carriers, adjuvants, etc.

In a preferred aspect of the invention, a sustained/controlled releaseH3 relaxin formulation was a selectively permeable outer barrier with adrug dispensing opening, and an inner H3 relaxin containing portiondesigned to deliver dosage of the H3 relaxin progressively diminishingat a predetermined rate (e.g. containing about 30 mg of H3 relaxin in amatrix for delivery of initially about 500 .mu.g per day diminishing asa rate of 10 .mu.g per day.

In another preferred aspect of the invention, a sustained/controlledrelease of H3 relaxin has a selectively permeable outer barrier with adrug dispensing opening, a first inner H3 containing portion designedfor steady state release of H3 relaxin at a therapeutically effectivedaily dosage (e.g. containing about 50 mg of H3 relaxin in a matrix forcontinuous delivery of about 500 .mu.g per day), and a second inner H3relaxin a portion designed to deliver a dosage of H3 relaxinprogressively diminishing at a predetermined rate (e.g. containing about3 mg of H3 relaxin in a matrix for delivery of initially about 500 .mu.gper day diminishing at a rate of 50 .mu.g per day) commencing uponexhaustion of the H3 relaxin from the first inner portion.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,preferably about 0.5% to 50%, by weight of H3 relaxin, either alone orin combination with H3 relaxin, the remainder being suitablepharmaceutical excipients, carriers, etc. Actual methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthis art; for example, see Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., 15th Edition, 1975.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration. Parenteraladministration is generally characterized by injection, eithersubcutaneously, intradermally, intramuscularly or intravenously,preferably subcutaneously. Injectable can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol or the like. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, solubility enhancers, and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, cyclodextrins, and the like.

A more recently devised approach for parenteral administration employsthe implantation of a slow-release or sustained-release system, suchthat a constant level of dosage is maintained. See, e.g., U.S. Pat. No.3,710,795.

Alternately, one may administer the H3 in a local rather than systemicmanner, for example, via injection of the compound directly into a solidtumor, often in a depot or sustained release formulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with tumor-specific antibody.The liposomes will be targeted to and taken up selectively by the tumor.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containiing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. The cosolventsystem may be the VPD co-solvent system. VPD is a solution of 3% w/vbenzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and65% w/v polyethylene glycol 300, made up to volume in absolute ethanol.The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5%dextrose in water solution. This co-solvent system dissolves hydrophobiccompounds well, and itself produces low toxicity upon systemicadministration. Naturally, the proportions of a co-solvent system may bevaried considerably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of polysorbate 80; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various of sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Formulations comprising human H3 relaxin may also be administered to therespiratory tract as a nasal or pulmonary inhalation aerosol or solutionfor a nebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose, or with otherpharmaceutically acceptable excipients. In such a case, the particles ofthe formulation may advantageously have diameters of less than 50microns, preferably less than 10 microns. See, e.g., U.S. Pat. No.5,364,838, which discloses a method of administration for insulin thatcan be adapted for the administration of H3 relaxin.

H3 relaxin for treatment of such disorders such as alopecia, may also beadministered topically in a formulation adapted for application to thescalp, such as a shampoo (e.g., as disclosed in U.S. Pat. No.4,938,953,adapted according to methods known by those skilled in the art, asnecessary for the inclusion of protein ingredients) or a gel (e.g., asdisclosed in allowed U.S. Ser. No. 08/050,745) optionally with increasedH3 relaxin concentrations to facilitate absorption.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

Various aspects of the invention will be described with reference to thefollowing non-limiting examples.

In the examples which follow rats are used as an experimental model tomap H3 relaxin expression at the mRNA and protein level in the ratbrain, this allowing human brain mapping. In this regard, the rat brainis a standard comparative anatomical model of the human brain (Goto etal (2001) The Journal of Comparative Neurology 438: 86-122).

EXAMPLE 1 Nucleotide Sequence Identification, Characterisation,Purification and Manipulation

Tissue RNA/DNA Extraction and RT-PCR-Human genomic DNA was extractedfrom human CL using standard protocols (Sambrook et al 1989) InMolecular Cloning. A Laboratory Manual, 2nd Ed., Cold Spring HarbourLaboratory Press, N.Y.). Human CL and mouse tissues were finely diced inthe presence of liquid nitrogen and immediately homogenized with RNAWizreagent (Ambion Inc., Austin, Tex.), and the RNA extracted according tothe manufacturer's instructions. Total RNA (5 μg) from each sample wasused for the reverse transcription (RT) reaction, which was preformedusing the Superscript II RT-PCR kit (Gibco-BRL, Rockville, Md.) in a 20μl volume according to the manufacturer's instructions. A 50 μl reactioncontaining 100 ng of primers and 150 ng of the cDNA template was usedfor all PCR reactions. Mouse tissues were screened for M3 relaxinexpression using specific forward [5′ TGCGGAGGCTCACGATGGCGC 3′] andreverse [5′ GACAGCAGCTTGCAGGCACGG 3′] primers, which generated a 319-bpproduct. Mouse relaxin (M1) expression was determined using a specificforward [5′ GTGAATATGCCCGTGAATTGATC 3′] and reverse [5′AGCGTCGTATCGAAAGGCTCT 3′] primer based on the published sequence (Evanset al (1993) J. Mol. Endocrinol. 10, 15-23), generating a 150-bpproduct.

Human CL cDNA was used in RT-PCR reactions with specific primers for H3relaxin, forward 1 [5′ ACGTTCAAAGCGTCTCCGTCC 3′], forward 2 [5′CGGTGGAGACGATCAGACATC 3′] and reverse [5′ ATGGCAGGACTGGGGCATTGG 3′],generating products of 504- and 310-bp for forward 1/reverse and forward2/reverse, respectively. All primer combinations we subsequently show tocross the single introns in the mouse and human relaxin sequences,respectively, so as to control for genomic DNA contamination. In allexperiments GAPDH forward [5′ TGATGACATCAAGAAGGTGG 3′] and reverse [5′TTTCTTACTCCTTGGAGGCC 3′] primers generating a product of 246-bp wereused in separate PCR reactions to control for quality, and equivalentloading of the cDNA. M3 relaxin expression by RT-PCR was performed oncDNA samples extracted from at least two animals, although the resultsfrom only one representative experiment are shown. The mouse PCRreactions were completed in a Perkin Elmer Gene Amplifier using thefollowing (touch-down) annealing temperatures: 64° C. (2 cycles), 63° C.(2 cycles), 62° C. (2 cycles), 61° C. (2 cycles), 60° C. (32 cycles). H3relaxin expression in human CL cDNA was performed by RT-PCR at thefollowing annealing temperatures: 60° C. (2 cycles), 59° C. (2 cycles),58° C. (2 cycles), 57° C. (2 cycles), 56° C. (32 cycles). Aliquots ofthe PCR products were electrophoresed on 2% (w/v) agarose gels stainedwith ethidium bromide and photographed. Mouse tissue samples weretransfered to Hybond NX membranes (Amersham International, Aylesbury,UK) for Southern blot analysis.

An additional PCR reaction was performed using mouse brain and ovariancDNA using the reverse M3 primer (above) and a forward primer from infront of the ATG start codon (5′ GGG TCGCAGGCATCTCAACTG 3′). Theresulting product contained the full H3 relaxin coding sequence. PCR wasperformed as above, but with the following annealing temperatures: 60°C. (2 cycles), 59° C. (2 cycles), 58° C. (2 cycles), 57° C. (2 cycles),56° C. (32 cycles). To generate a specific H3 relaxin cDNA probe for³²P-labeling and to utilize it for subsequent probing of a human multitissue array, RT-PCR was performed on human genomic DNA (50 ng).Specific forward (5′ CGGATGCAGATGCTGATGAAG 3′) and reverse (5′GTGCCTGAGCCCACAGTGCCT 3′) primers from the exon II sequence of the H3relaxin gene were used at the following annealing temperatures: 60° C.(2 cycles), 59° C. (2 cycles), 58° C. (2 cycles), 56° C. (2 cycles), 54°C. (32 cycles). These products as well as the mouse PCR productdescribed above, were separated on 2% agarose gels. Bands were detectedof the expected size under UV light (mouse 319-bp, 478-bp; human374-bp), excised and eluted from the gel using the Ultraclean TM 15 DNApurification kit (Geneworks Pty Ltd, Adelaide, Australia). The bandswere subsequently subcloned into the pGEM-T vector (Promega, Madison,Wis.) and multiple subclones were then sequenced on both strands usingthe ABI PRISM 377 automatic DNA sequencer, according to themanufacturers instructions (Applied Biosystems, Melbourne, Australia).

Southern Blot Analysis—PCR products on membranes were hybridized againstspecific internal oligonucleotide primers for the M1 relaxin (5′CAAGCAGAGCTGGCTCCTCCTGGCT CAAAGCCAATCTTC 3′) and M3 relaxin (5′AATTTGGCTCTTGCTACAGCCCCACTCG CAGCAACTGCT 3′) cDNA sequences, which hadbeen labeled using T4 polynucleotide kinase and [γ-³²P] ATP.Hybridization was performed at 55° C. overnight in 5×SSC (1×SSC; 0.15 MNaCl, 15 mM sodium citrate, pH 7), 5×Denhardts, 1% SDS and 100 μg/mlsonicated herring sperm. Membranes were washed three times for 5 min in2×SSC, 0.1% SDS at room temperature followed by a 30 min wash at 55° C.in 0.1×SSC, 0.1% SDS, before being exposed to BioMAX MR film (EastmanKodak Co., Rochester, N.Y.) for 24 h at room temperature.

Northern Blot Analysis—To further examine the expression of M3 relaxinmRNA, total RNA (5-25 μg) from the heart, brain, lung, thymus and spleenof male mice, and ovary, endometrium, myometrium, cervix and vagina offemale mice pooled from day 7.5, 10.5 and 18.5 of pregnancy, were run onstandard MOPS/formaldehyde gels. RNA was then transferred to optimizedHybond-NX membranes and probed for M3 relaxin mRNA with a ³²P-labeledprobe that corresponded to the 319-bp PCR product, generated by specificprimers to M3 relaxin (see above). This product was labeled with[α-³²P]dCTP using the specific reverse primer (above) and T7 polymeraseas previously described (31). The membrane was hybridized at 65° C.overnight in 0.25M NaH₂PO₄, pH 7.2, 1 mM EDTA, 20% SDS, followed bythree washes for 5 min in 2×SSC, 0.1% SDS at room temperature, andfinally a 30 min wash at 65° C. in 0.1×SSC, 0.1% SDS. Membranes werefirst exposed to a phosphoimager plate for 48 h at room temperaturebefore being analysed in a FujiX 2000 Phosphoimager (Fuji Photo Company,Japan), and then exposed to BioMAX MS film (Integrated Sciences,Melbourne, Australia) together with a Hyperscreen (Amersham Pharmacia,Sydney, Australia) at −80° C. In separate experiments, total RNA (200μg) from the brain, spleen, liver and testes was purified to poly-A RNAusing an mRNA purification kit (Amersham Pharmacia), and Northernblotting performed as described above. A human multiple tissueexpression array (CLONTECH laboratories, Palo Alto, Calif.) washybridized with a ³²P-labeled H3 relaxin specific probe according to themanufacturers recommendations. The 374-bp fragment of the H3 relaxinsequence isolated from genomic DNA was labeled with [α-³²P]dCTP usingthe H3 relaxin specific reverse primer (described above), and T7polymerase (Bathgate et al (1999) Biol. Reprod. 61, 1090-1098). Themembrane was exposed to a phosphoimager plate and BioMAX film asdescribed above.

In Situ Hybridization Histochemistry—Coronal sections (14 μm) were cuton a cryostat at −16° C. and mounted on silane-coated slides. Sectionswere delipidated in chloroform for 10 min, rinsed and stored in 100%ethanol at 4° C. Three oligonucleotides (39 mers) [5′ GGTGGTCTGTATTGGCTTCTCCATCAGCGAAGAAGTCCC 3]′; [5′ AATTTGGCTCTTGCTACAGCCCCACTCGCACGAACTGCT 3′] and [5′ TAAGGAGACAGTGGACCCCTTGGTGCCTCGCCTGT AGGA 3′],of the M3 relaxin mRNA sequence, and three oligonucleotides to [5′GCACATCCGAATGAATCCGTCCATCCACTCCTCCGAGAC 3′], [5′ CAAGCAGAGCTGGCTCCTCCTGGCTCAAAGCCAATCTTC 3′] and [5′ GTTGTAGCTCTGGGAGCGAGGCCTGAGCCTCAGACAGTA 3′] of the previously known M1 relaxin sequence (Evanset al (1993) J. Mol. Endocrinol. 10, 15-23) were prepared commercially(Geneworks Pty Ltd). Probes were labeled with [α-³⁵S]dATP (1200 Ci/mmol;NEN, AMRAD-Biotech, Melbourne, Australia) to a specific activity of 1×10⁹ d.p.m./μg using terminal deoxynucleotidyl transferase (RocheDiagnostics; Wisden et al (1994) In In Situ Hybridization Protocols forthe Brain (Wisden, W. and Morris, B. J. eds), pp 9-34, Academic Press,London). Screening of the sequences used against gene sequence databases(Celera, EMBL and Genbank; NCBI/NIH Blast Service) revealed homologyonly with the appropriate M1 and M3 relaxin mRNAs.

Sections were incubated overnight at 42° C. with multiple ³⁵S-labeledprobes (30 fmol each probe/slide) in hybridization buffer containing 50%formamide, 4×SSC, 10% dextran sulphate and 0.2 M dithiothreitol. Slideswere washed in 1×SSC at 55° C. for 1 h, rinsed in 0.1×SSC, thendehydrated before being apposed to Kodak BioMAX MR for 10 d.

The authenticity of the hybridization was confirmed by the demonstrationthat the signal could be successfully blocked in all areas by theaddition of a 100-fold excess of unlabeled probes to the hybridizationbuffer, except those that corresponded to non-specific or backgroundhybridization (data not shown). In addition, three oligonucleotideprobes were used that were complementary to different, non-overlappingregions of the M3 relaxin gene sequence.

Human Relaxin (H3) Studies:

Solid Phase Synthesis—A putative peptide sequence encoded by the H3 genewas assembled by solid phase synthesis procedures based on the predictedsignal peptide and proteolytic enzyme cleavage sites between the signalpeptide and the B-chain, and the B/C and C/A chain junctions of the H3relaxin prohormone (see Results for details). For ease of synthesis wechose to prepare the A- and B-peptides as their carboxyl-terminal amidederivatives. Selectively S-protected A- and B-chains were synthesized ona 0.1 mmol scale by the continuous flow Fmoc solid-phase method aspreviously described Dawson et al (1999) J. Pept. Res. 53, 542-547.Selective S-protection was afforded for the following cysteine residues:trityl (Trt) for A^(10,15) and B²², tert-butyl for A²⁴, andacetamidomethyl (Acm) for A¹¹ and B¹⁰ (see FIG. 2A for numbering ofamino acid residues).

On completion of the syntheses, the S-protected A- and B-chains werecleaved from the solid supports and simultaneously sidechain deprotectedby treatment with TFA in the presence of scavengers. Selective disulfidebond formation was achieved essentially as described for the synthesisof bombyxin Maruyama et al (1992) J. Prot. Chem. 11, 1-12.

Peptide Charactenzation—Peptides were quantitated by duplicate aminoacid analysis of 24 h acid hydrolyzates on a GBC automatic analyser(Melbourne, Australia). MALDITOF mass spectrometry (MS) was performed inthe linear mode at 19.5 kv on a Bruker Biflex instrument (Bremen,Germany) equipped with delayed ion extraction.

Other Relaxin and Insulin Peptides—Human INSL3 was synthesized using thesame methodology used for ovine INSL3 (Dawson et al (1999) J. Pept. Res.53, 542-547), and was characterized by MS and amino acid analysis asoutlined above. H1 relaxin was synthesized previously (Wade et al (1996)Biomed. Pept. Prot. Nucl. Acids 2, 27-32), recombinant H2 relaxin was agift from the Connetics Corporation (Palo Alto, Calif.) and bovineinsulin was purchased from Roche Diagnostics (Sydney, Australia).

THP-1 Cell Bioassay—The ability of H3 relaxin to induce cAMP productionin the human monocytic cell line (THP-1) was compared to H1 and H2relaxin following the procedure of Parsell and colleagues (Parsell et al(1996) J. Biol. Chem. 271, 27936-27941), with the followingmodifications; THP-1 cells which had been viability tested using TrypanBlue were resuspended in media, and transferred to a 96 well plate at adensity of 60,000 cells/well. Peptides (H1, H2, H3 relaxin, human INSL3and bovine insulin) were added to the wells together with 1 μM forskolinand 50 μM isobutylmethylxanthine (IBMX) in RPMI media, and incubated at37° C. for 30 min. The plate was then briefly centrifuged, the mediaremoved and the cells resuspended in lysis buffer. cAMP levels weremeasured in the lysates using the cAMP Biotrak EIA system (AmershamInternational, Aylesbury, UK). The results are expressed as the maximumrelaxin response (%/O) in comparison to the maximum stimulation of cAMPachieved with H2 relaxin. Data represent the mean±SEM of threeexperiments performed in quadruplicate, and are plotted using PRISM(Graphpad Inc., San Diego, Calif.).

THP-1 Cell Binding Assay—THP-1 cells were spun down and resuspended inbinding buffer (20 mM HEPES, 50 mM NaCl, 1.5 mM CaCl₂, 1% BSA, 0.1 mg/mllysine, 0.01% NaN₄, pH 7.5) (Parsell et al (1996) J. Biol. Chem. 271,27936-27941) to give 2×10⁶ cells/well in a 96-well plate. The cells wereincubated in binding buffer with ³³P-labeled H2 (B33) relaxin (100 pM:labeled as previously described (Tan et al (1999) Br. J. Pharmacol. 127,91-98) at 25° C. for 90 min in the absence or presence of increasingconcentrations of unlabeled H1, H2 and H3 relaxin (100 pM to 30 nM).Non-specific binding was defined with H2 relaxin (1 μM. Cells wereharvested using a Packard 96-well plate cell harvester and Whatman GF/Cglass fibre filters treated with 0.5% polyethylenimine. The filters werewashed three times with modified binding buffer (20 mM HEPES, 50 mMNaCl, 1.5 mM CaCl₂), dried in a 37° C. oven, and the radioactivitycounted by liquid scintillation spectrometry (TopCount™, CanberraPackard, Australia).

Antibody Crossreactivity—The ability of well characterized human relaxinantibodies to recognize H3 relaxin was tested in comparison to H1 and H2relaxins by radioinmmunoassay. Briefly, goat anti-H2 relaxin (Lucas etal (1989) J. Endocrinol. 120, 449-57) was coated onto 96 well ELISAplates (Disposable Products, Adelaide, Australia) at a dilution of1:1000 with 0.05M sodium carbonate buffer at 4° C. overnight. Afterwashing twice with PBS-T (phosphate buffered saline; 0.05% Tween 20, pH7.4) dilutions of human relaxin peptides dissolved in 50 μl of assaybuffer (1% BSA in PBS-T) were added together with 50,000 cpm¹²⁵I-labeled relaxin, in 50 μl of assay buffer. H2 relaxin was¹²⁵I-labeled and purified by HPLC (Palejwala et al (1998) Endocrinology139, 1208-1212). After an overnight incubation at 4° C. the plates werewashed twice with PBS-T. The antibody-bound-¹²⁵I-labeled H2 relaxin wascollected by the addition of 1M NaOH and decanted into tubes forcounting on a Packard 5010 gamma counter (Canberra Packard). Experimentswere performed at least twice and similar results obtained. Data wasplotted as the mean±SEM from one representative experiment performed intriplicate and plotted using PRISM.

Mouse Relaxin (M3) Studies:

Animals—All male and female mice used in these studies were age-matchedand had the same background (C57BLK6J). Animals were housed in acontrolled environment and maintained on a 14 h light, 10 h darkschedule with access to rodent lab chow (Barastock Stockfeeds,Melbourne, Australia) and water. Female mice (3.5 months old) were matedand pregnancy timed from the identification of the vaginal plug. At day7.5, 10.5 and 18.5 of pregnancy, mice were sacrificed for tissuecollection. Tissues were also collected from non-pregnant female andmale mice (4 months old). These experiments were approved by the HowardFlorey Institute's Animal Experimental Ethics Committee, which adheresto the Australian Code of Practice for the care and use of laboratoryanimals for scientific purposes.

Tissue Collection—Animals were killed with an overdose of Isofluorane(Abbott Australasia Pty Ltd, Sydney, Australia). The brain, heart,thymus, spleen, lung, liver, kidneys, skin and gut were collected alongwith the reproductive organs from female (ovary, endometrium,myometrium, cervix, vagina; n=2 for each pregnancy stage) and male(testes, epididymis, prostate; n=3) mice. From additional animals, malebrains (n=3) were dissected into specific regions including thehypothalamus, cortex, hippocampus, thalamus, medulla and cerebellum, andimmediately placed in liquid nitrogen and stored at −80° C. until usedfor RNA preparation. Female brains (n=3) were collected and immediatelyfrozen over dry ice for in situ hybridization histochemistry (Burazin etal (2001) J. Neuroendocrinol. 13, 358-370). Human CL from women in earlypregnancy undergoing surgery for ectopic pregnancies were utilized withthe approval of the Howard Florey Institute Human Ethics Committee andthe written consent of the patients.

EXAMPLE 2 Human H3 Relaxin Genes in the Human and Mouse

Both H3 relaxin sequences in the human and mouse contain featuresrepresentative of functional genes (FIG. 1A human; 1B mouse). Eachcontain a putative TATA box for initiation of transcription 65, and 59bp, upstream of putative ATG start codons for human and mouse,respectively. A polyadenylation signal is present in the 3′ untranslatedregion of both genes, in a position 582 and 448 bp downstream from aninframe TAG stop codon for the human, and mouse genes respectively. Asingle intron interrupts the coding region in an identical position inthe sequence of both genes, corresponding to a similar position to thatof other relaxin and insulin family members (Hudson et al (1983) Nature301, 628-631; Evans et al (1993) J. Mol. Endocrinol. 10, 15-23; Ivell, R(1997) Rev. Reprod. 2, 133-138). The H3 relaxin gene is localized onchromosome 19 at 19p13.3, whereas the mouse gene is located onchromosome 8 at 8C2. The derived coding regions of the H3 and M3 relaxingenes were 142, and 141, amino acids, respectively.

The cysteine residues necessary for disulphide bond formation areretained in the correct positions, together with conserved glycineresidues necessary for flexibility around the cysteine linkages(Büllesbach et al (2000) Int. J. Pept. Prot. Res. 46, 238-243). Mostimportantly, the residues demonstrated to be essential for relaxinreceptor binding in the core of the B-chain (R—X—X—X—R—X—X—I)(Büllesbach et al (2000) J. Biol. Chem. 275, 35276-35280), have beenretained in both the human and mouse sequences. Therefore, although thehuman sequence most closely resembles the hINSL5 peptide sequence ondirect amino acid homology, the presence of this binding motif indicatesthat the peptide is more like a relaxin peptide. Interestingly, the M3relaxin A-chain conforms to the cysteine pattern of family members,whereas the previously characterized M1 relaxin sequence contains anextra tyrosine residue before the final cysteine residue (FIG. 2A).

The H3 (human H3) and M3 (mouse “3” relaxin) sequences share greaterthan 70% homology in the coding region at the nucleotide level. However,the homology is most striking in the derived amino acid sequence. Bothderived pro-hormone sequences contain a typical signal sequence afterthe ATG start codon which is likely to be cleaved at an identicalposition between alanine and arginine in both the human and mousepeptides (Nielsen et al (1997) Prot. Engineer. 10, 1-6). Thearginine-arginine pair of basic amino acids at the B/C junction foundwith other members of the relaxin family strongly suggests cleavagebetween tryptophan and arginine. Similarly, cleavage at the C/A junctionis most likely to occur between the arginine and aspartic acid asindicated in FIGS. 1A and 1B, as this corresponds to a weak furin(proprotein convertase) cleavage site (Nakayama, K. (1997) Biochem. J.327, 625-635. Therefore, it is believed that both H3 and M3 relaxinscomprise a B-chain of 27 amino acids, a C-peptide of 66 amino acids andan A-chain of 24 amino acids.

A comparison of the A- and B-chain sequences of H3 and M3 relaxin withH1, H2 and M1 relaxin is outlined in FIG. 2A. There are only two aminoacid differences in both the A- and B-chains between the M3 and H3sequences, of which three of these changes are conserved. In contrast,the homology between M1 and H2 relaxin is only 42% and 45% in the A-,and B-, chains respectively. Furthermore, other than the key coreelements in the B-chain and the key structural elements in the A-chain,there is very little homology between H2 and H3 relaxin, and between M1and M3 relaxin. Interestingly, H3 and M3 relaxin show high homology ofthe C-peptide domain (73%), compared with less than 20% homology in thisregion of other insulin/relaxin family members. The C-peptide lengths ofH3 and M3 relaxin are 65, and 66 amino acids, respectively, and are muchshorter than that of other relaxins (102 amino acids for H1 and H2 and99 amino acids for M1 relaxin). The C-peptide chain length and sequencehomology is most similar to INSL5 (24%).

The full length amino acid sequences of the two genes were aligned toother members of the insulin/relaxin family and a phylogenetic treegenerated (FIG. 2B). Additionally, the H3 and M3 relaxin sequences aregrouped under a separate branch, indicating that the evolution of theseparticular relaxins diverged from other relaxins early in evolution.This was also the case for INSL5 within this analysis whichinterestingly shares closest primary structural similarity to H3relaxin.

EXAMPLE 3 Peptide Synthesis

H3 relaxin was prepared by solid phase synthesis in low overall yield(0.7%). MALDTOF MS showed a single product with an MH⁺of 5,494.7(theoretical value: 5,497.5). Amino acid analysis also confirmed itscorrect composition.

Chemical Synthesis of Human Relaxin H3 [hRlx-3 A(1-24) amide-B(l-27)amide

Selectively S-protected A- and B-chains representing the amino acidsequence of the separate H3 relaxin peptide chains, were synthesized bythe continuous flow 9-fluorenyl methoxycarbonyl (Fmoc) solid-phasemethod using the general procedures described in Atherton, E andSheppard, R C. (Solid Phase Peptide Synthesis. IRL Press at OxfordUniversity Press, Oxford, United Kingdom, 1989). Both peptides wereprepared on a 0.1 mmol scale as peptide-carboxyl terminal amides usingFmoc peptide amide linker polyethylene glycol polystyrene(Fmoc-PAL-PEG-PS) supports (Applied Biosystems). For the A-chainassembly, four-fold excesses of Fmoc-amino acids (Auspep, Melbourne,Australia) were activated by 1,3-diisopropylcarbodiimide (DIC) and1-hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), whereas duringthe B-chain synthesis each residue was activated by2-(1H-benzotrazole-1-yl)-1,1,3,3-tetramethyluronium hexaflurophosphate(HBTU) and diisopropylethylamine (DIEA) in DMF. N^(α)—Fmoc deprotectionfor both chain assemblies was with 20% piperidine in DMF. Couplings weregenerally of 30 minutes duration, with the exception of double couplingsand extended times for A-chain residues Ser^(7,8,21) and all cysteines,and double couplings of B-chain residues Arg^(1,12,16), Ala^(2,3,17) andCys¹⁰. Side chain protection was afforded by tert-butyl esters andethers for Asp, Glu, Thr and Ser, butoxycarbonyl (Boc) for Lys and Trp,2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl (Pbf) for Arg and theamide bond protection N^(α)-(2-Fmoc-oxy-4-methoxybenzyl) [FmocHmb] forB-chain Gly¹¹. Selective S-protection was afforded for the followingcysteine residues: trityl (Trt) for Cys^(10,15) in the A-chain and Cys²²in the B-chain, tert-butyl (tBu) for Cys²⁴ in the A-chain, andacetamidomethyl (Acm) for A-chain Cys¹¹ and B-chain Cys¹⁰.

(i) Synthesis of Human Relaxin H3. A-chain [Cys¹¹(Acm),Cys²⁴(tBu)](1-24) amide [1]

On completion of the synthesis, the protected A-chain resin was treatedat room temperature for 2.5 hours with 95% trifluoroacetic acid(TFA)/2.5% ethanedithiol (EDT)/2.5% H₂O plus 4 drops triethylsilane, toaid the quenching of thiols. TFA was removed to a minimum volume under astream of nitrogen and precipitated twice from chilled diethyl ether.The precipitate was then dissolved in 0.1% aq. TFA and lyophilized. Thecrude S-reduced [thiol-Cys^(10,15), Cys¹¹(Acm), Cys²⁴(t-Bu)] A-chain wasdirectly subjected to air oxidation in 0.1M Gly-NaOH, pH 8.3, for 4hours at room temperature. Analytical reverse-phase high performanceliquid chromatography (RP-HPLC) monitoring confirmed the completeness ofthe intramolecular disulfide bond formation, after which several dropsof neat TFA were added and the crude oxidized material directlylyophilized.

(ii) Synthesis of Human Relaxin H3, B-chain [Cys¹⁰(Acm)](1-27) amide [2]

On completion of the synthesis, the protected B-chain resin was treatedat room temperature for 2.5 hours with 82.5% TFA/5% phenol/5% H₂0/5%thioanisole/2.5% ethanedithiol plus 4 drops of triethylsilane, to aidthe quenching of thiols. TFA was removed to a mininmum volume under astream of nitrogen and precipitated twice from chilled diethyl ether.The precipitate was then dissolved in 0.1% aq. TFA and lyophilized. Thecrude B-chain was then purified by RP-HPLC as described below.

(iii) Synthesis of Human Relaxin H3, A-chain [Cys¹¹(Acm),Cys²⁴(Pyr)](1-24) amide [3]

25 mg of peptide 1 (9.65 μmol) and 35 mg (158.86 μmol) 2,2′-dipyridyldisulfide (DPDS) were dissolved together in 4.5 ml TFA and 0.5 mlthioanisole and the resulting solution then chilled. To this was added 5ml trifluoromethanesulfonic acid (TFMSA)/TFA (1:5 v/v) and the wholemixture allowed to stir at ≦0° C. for 30 mins. The [Cys¹¹(Acm),Cys²⁴Pyr)] A-chain amide peptide was precipitated from cold ether andthe pellet obtained on centrifugation then suspended in 6M guanidinehydrochloride (GdHCl), pH 8.0, and purified by RP-HPLC. (Yield peptide3: 4%). (Alternative to RP-HPLC purification, peptide 3 was desalted ona Sephadex G-25 gel filtration column in 20% aq acetic acid).

(iv) Synthesis of Human Relaxin H3, A[Cys¹¹(Acm)](1-24)amide-B[Cys¹⁰(Acm)](1-27) amide [4]

Purified A-chain peptide 3 (1.0 mg, 0.38 μmol) and purified B-chainpeptide 2 (1.2 mg, 0.38 μmol) were dissolved separately in 1.0 ml and0.5 ml 0.1M NH₄HCO₃ respectively. The B-chain solution was then slowlyadded to A-chain and the reaction mixture was stirred vigorously at roomtemperature for 30 min. The solution was acidified with 0.5 ml glacialacetic acid and then subjected to RP-HPLC, as detailed below, to isolatethe bis-disulfide bonded chain combined product. (Alternative to RP-HPLCpurification, the resulting A/B product, peptide 4, was desalted on aSephadex G-25 gel filtration column in 20% aq acetic acid).

(An alternative method for chain combination which improves B-chainsolubility, is as follows: peptide 3 and purified [Cys¹⁰(Acm)] B-chainwere dissolved separately, at a concentration of 1.0 ml/mg, in 8M GdHCl,pH 4.5 buffer. The B-chain solution was then slowly added to A-chain andthe reaction mixture was stirred vigorously at 37° C. for 24 hours).

(v) Synthesis of Human Relaxin H3 [hRlx-3A(1-24) amide-B(1-27) amide

All of the purified 4 peptide was used to form the third and finaldisulfide bond (assuming 100% recovery, estimated at 0.39 μmol). Thepeptide was dissolved in a solution of 80 mM HCl and acetic acid. 20 mMiodine in 95% aqueous acetic acid was then added dropwise (25 equivs ofiodine per Acm group). The reaction was performed for 1 hour in the darkat room temperature after which excess oxidant was quenched with 20 mMaqueous ascorbic acid. Purification of the relaxin was by RP-HPLC, witha final yield, relative to peptide 3 starting material, of 0.74%.

Purification

The separate crude chains and intermediate peptides were purified byRP-HPLC, using a Waters 600 multisolvent delivery system connected to amodel 996 photodiode array detector. A 10×250 mm Vydac 218 TP columnpacked with C₄ silica gel (330 A pore size, 10 μm particle size) wasused. The peptides were eluted with a solvent system of (A) 0.1% aq. TFA(v/v) and (B) 0.1% TFA in acetonitrile (v/v) in a linear gradient mode(25-50% B over 30 minutes). The target fractions were collected andidentified by matrix-assisted laser desorption ionization massspectrometry (MALDI-TOF MS) and lyophilized.

Peptide Characterisation

Peptide quantitation was by duplicate amino acid analysis of 24 hr acidhydrolyzates on a GBC automatic analyser (Melbourne, Aust). MALDITOF MSwas performed in the linear mode at 19.5 kv on a Bruker Biflexinstrument (Bremen, Germany) equipped with delayed ion extraction.

EXAMPLE 4 Relaxin Biological Activity

Demonstration of Relaxin Activity of Synthetic H3 Relaxin-Synthetic H3relaxin C-terminal amide derivatives were tested for relaxin activity ina relaxin receptor expressing cell line, THP-1 (Parsell et al (1996) J.Biol. Chem. 271, 27936-27941). H2 relaxin produces a dose dependentincrease in cAMP production from these cells (FIG. 3A). Synthetic H3relaxin also stimulated a dose dependent increase in cAMP(pEC₅₀=8.68±0.08 [2.11 nM]; n=3), albeit with slightly lower activitythan H1 (pEC₅₀=9.10±0.05 [0.794 nM]; n=3) and H2 (pEC₅₀=9.67±0.11 [0.214nM; n=3) relaxin. The specificity of this response was demonstrated bythe inability of bovine insulin (bINSL), or human insulin 3 (hINSL3), tostimulate cAMP responses at doses up to 1 μM.

Synthetic H3 relaxin was also tested for its ability to compete for³³P-labeled H2 relaxin binding to relaxin binding sites in THP-1 cells(FIG. 3B), with an affinity (pK_(i)=7.5±0.16; n=3) lower than that of H2(pK_(i)=8.74+0.11; n=11) and H1 (pK_(i)=8.9+0.11; n=7) relaxin.Nevertheless, these data provide definitive evidence that the syntheticH3 relaxin peptide binds to, and elicits a second messenger response bystimulating human relaxin receptors.

Ability of a Well Characterized H2 Relaxin Antibody to Recognize H3Relaxin—The ability of a well characterized anti-H2 relaxin antibody torecognize H1 and H3 relaxin was tested by radioimmunoassay. As shown inFIG. 4, H2 relaxin was able to displace ¹²⁵I-labeled H2 relaxin bindingto the anti-H2 relaxin antibody with high specificity. In contrast, H1and H3 relaxin showed poor cross reactivity with the antisera asdetermined by their poor ability to displace ¹²⁵I-labeled H2 relaxinbinding. Furthermore, the non-parallellism of the displacement curvesindicates that not all the antibody epitopes are recognized by the twopeptides.

EXAMPLE 5 H3 Relaxin Expression

Relaxin Gene Expression in the Mouse—The expression of M3 relaxin mRNAwas compared to M1 relaxin mRNA expression using southern blotting ofRT-PCR products. Although this technique is only semi-quantitative, itenabled us to determine the potential sites of expression of M3 relaxincompared to M1 relaxin. The results of a representative experiment andduplicate experiments gave identical results. M3 relaxin mRNA wasexpressed in a number of tissues in C57BLK6J mice where M1 relaxin wasfound, but the pattern of expression, between the two mouse relaxins wasdifferent. In male non-reproductive tissues, highest levels of M1relaxin expression were seen in the brain, moderate levels in thethymus, heart and kidney, lower levels in the lung, spleen and skin,with no expression seen in the gut. Interestingly, M3 relaxin expressionwas detected at highest levels in brain, however, it was expressed atmoderate levels in the thymus, lung and spleen, only at very low levelsin the heart and liver, and not at all in the kidney, skin and gut.Female mice showed an almost identical pattern of expression for bothgenes in these tissues. In male reproductive tissues M3 relaxin mRNA wassignificantly expressed only in the testis whereas, M1 relaxin mRNA wasdetected in the testis, epididymis and prostate. Both relaxins were alsodetected in female reproductive organs in the mammary gland, ovaries ofnon-pregnant, pregnant and lactating mice, and the endometrium andmyometrium of pregnant mice. Significant expression of M3 relaxin mRNAwas observed in all ovarian stages, while M1 relaxin expression washigher in ovaries of late gestation compared to ovaries fromnon-pregnant and lactating mice. High levels of M3 relaxin mRNA weredetected in the brain and further analysis of this tissue revealed thatboth relaxins were expressed in several distinct regions. While M1relaxin mRNA was consistently expressed in the hypothalamus,hippocampus, cortex, thalamus, pons/medulla and cerebellum, M3 relaxinmRNA was found to be highly expressed in the thalamus and pons/medulla,thus suggesting, that the two relaxins may play distinct roles in themouse.

Northern Analysis—Tissues in which M3 relaxin mRNA was positivelyidentified by RT-PCR and Southern blot analysis, were further examinedby Northern blotting. Total RNA (5-25 μg) from the heart, brain, lung,thymus, spleen, ovary, endometrium, myometrium, cervix and vagina wereinitially probed with a ³²P-labeled M3 relaxin specific probe, but nospecific hybridizing bands were found in any tissue. Poly-A RNA from thebrain (15 μg), spleen (5 μg), liver (5 μg) and testis (25 μg) were thenanalyzed and a specific ˜1.2-kb hybridizing band was identified in thebrain, consistent with M3 relaxin expression detected by RT-PCR andSouthern blot analysis. The obtained transcript size was consistent withthe predicted size based on the M3 relaxin transcript sequence (˜1 kb)plus a poly-A tail (˜200-bp).

Expression of H3 Relaxin in Human Tissues—A Clonetech Multi TissueExpression Array was used to examine sites of expression of H3 relaxinin human tissues. The array contained normalized poly-A RNA (50-750 ng)from 76 different human tissues including 8 different control RNAs andDNAs, spotted onto a nylon membrane. The array was probed with a³²P-labeled 374-bp H3 relaxin specific gene fragment from the 3′ end ofthe H3 relaxin transcript, generated from genomic DNA. This DNA fragmentwas sequenced on both strands. Very weak hybridizing signals wereobserved in spleen, thymus, peripheral blood leukocytes, lymph node andtestis however, these signals were barely discernable above backgroundand hence, the data is not shown. RT-PCR was also performed on human CLfrom early pregnancy using two different primer combinations based onthe H3 relaxin sequence. No specific bands were observed in any PCRreaction even after changing the PCR conditions, whereas transcripts forH2 relaxin and GAPDH were easily amplified (data not shown), confirmingthe integrity of the cDNA.

Distribution of Relaxin mRNA in the Mouse Brain—Given the high levels ofM3 relaxin mRNA expression detected by RT-PCR and Northern blotting inthe brain, its distribution was further examined using in situhybridization histochemistry (Burazin et al (2001) J. Neuroendocrinol.13, 358-370. Multiple specific ³⁵S-labeled oligonucleotide probes wereutilized to determine the cellular distribution of M3 relaxin mRNAthroughout the rostro-caudal extent of the female C57BLK6J mouse brain.M3 relaxin mRNA was not widely detected throughout brain nuclei, but wasmost strongly detected in the pons/medulla (FIG. 7). The strongest levelof M3 relaxin mRNA was present in the pars ventromedialis of the dorsaltegmental nucleus. In addition, M3 relaxin mRNA was also detected,albeit at far lower levels, in the hippocampus and olfactory regions.Brain regions containing low levels of mRNA encoding M3 relaxin may nothave been detected in the current study due to sensitivity limitationsassociated with in situ hybridization histochemistry. The distributionof M3 relaxin mRNA in the brain differs from that of M1 relaxin mRNA, asno M1 relaxin mRNA was detected in the pars ventromedialis of the dorsaltegmental nucleus (data not shown).

EXAMPLE 6

Prorelaxin H3 cDNA sequences from human, mouse and rat are expressed inboth prokaryotic and eukaryotic cell systems using appropriateexpression transfer vectors.

These systems include appropriate mammalian host cells, other highereukaryotic cells including insect cells, plant cells and avian cells aswell as bacterial and yeast expression systems. Additionally, fusionprotein products of these three sequences are produced by linking aportion of a prokaryotic or eukaryotic protein characteristic of thehost cell. The fusion products facilitate the purification of theprotein product such that the fusion product may be subsequentlyremoved. All transfer vectors may also be modified by codonsubstitutions/deletions/additions with the modifications giving rise toshortened C peptide prorelaxins with B/C and C/A junction modificationsto facilitate the removal of the modified C peptide sequence.

Relaxin synthesis using shortened C peptide substitutions and B/C andC/A junction modifications are described in U.S. Pat. No. 5,759,807, andsuch methods may be used for the production of H3 relaxin.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and shouldnot be taken as an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

1. A method for the treatment of one or more of: vascular disease;treatment of arterial hypertension; diseases related to uncontrolled orabnormal collagen or fibronectin formation; kidney disease; psychiatricdisorders; depression or depressive disorders; neurologic orneurodegenerative diseases; disorders of learning, attention andmotivation; addictive disorders; movement and locomotor disorders;immunological disorders; breast disorders; endometrial disorders;endocrine disorders; delayed onset of labour, impaired cervicalripening, and prevention of prolonged labour due to fetal dystocia;sinus bradycardia; hair loss; alopecia; disorders of water balanceincluding impaired or inappropriate secretion of vasopressin; orplacental insufficiency; which comprises administering to a subject inneed of any such treatments a therapeutically effective amount of humanH3 relaxin, or an analogue thereof as herein defined, optionally inassociation with one or more pharmaceutically acceptable carriers and/diluents and/or excipients.
 2. A method according to claim 1 wherein theH3 relaxin or analogue thereof is human H3 relaxin, human H3 prorelaxin,human H3 preprorelaxin, or the constitutive A, B or C peptide chainsthereof.
 3. A method according to claim 2 wherein the human H3 relaxinor a human H3 relaxin analogue thereof comprises an A chain and a Bchain, the A chain having the amino acid sequence: Asp Val Leu Ala GlyLeu Ser Ser (SEQ ID NO: 4) 1               5 Ser Cys Cys Lys Trp Gly CysSer     10                  15 Lys Ser Glu Ile Ser Ser Leu Cys 20

or an amino acid sequence truncated by up to about 9 amino acids fromN-terminus, the B chain having the amino sequence: Arg Ala Ala Pro TyrGly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg1               5                   10                  15 Ala Val IlePhe Thr Cys Gly Gly Ser Arg Trp             20                  25

or an amino acid sequence truncated by up to 9 amino acids from theamino-terminus and/or up to about 5 amino acids from thecarboxyl-terminus, the A and B chains being linked by disulphide bondsbetween A11-B10 and A24-B22, and wherein the human H3 relaxin oranalogue thereof has relaxin bioactivity.
 4. A method according to claim1 wherein a human H3 relaxin analogue comprises a modified A chainand/or a modified B chain, the H3 relaxin A chain having the amino acidsequence: Asp Val Leu Ala Gly Leu Ser Ser Ser Cys Cys Lys Trp Gly CysSer (SEQ ID NO: 4)1               5                   10                  15 Lys Ser GluIle Ser Ser Leu Cys             20

wherein the carboxyl-terminus is an amide derivative and/or Lys atposition 12 is replaced with Glu, and/or Glu at position 19 is replacedwith Gln, the H3 relaxin B chain having the amino acid sequence: Arg AlaAla Pro Tyr Gly Val Arg Leu Cys Gly Arg Glu Phe Ile Arg (SEQ ID NO: 2)1               5                   10                  15 Ala Val IlePhe Thr Cys Gly Gly Ser Arg Trp             20                  25

wherein the carboxyl-terminus is an amide derivative, and/or Ala atposition 2 is replaced with Pro, and/or Arg at position 8 is replacedwith Lys, the A and B chains being linked by disulphide bonds betweenA11-B10 and A24-B22, and wherein the human H3 relaxin analogue hasrelaxin bioactivity.
 5. A method according to claim 1 which is a methodfor the treatment of arterial hypertension.
 6. A method according toclaim 1 which is a method for the treatment of peripheral vasculardisease including coronary artery disease, peripheral vascular disease,vasospasm including Raynaud's phenomenon, microvascular diseaseinvolving the central and peripheral nervous system, kidney, eye andother organs.
 7. A method according to claim 1 which is a method for thetreatment of kidney disease including vascular disease, interstitialfibrosis, glomerulosclerosis, or other kidney disorders.
 8. A methodaccording to claim 1 which is a method for the treatment of psychiatricdisorders including anxiety states including panic attack, agoraphobia,global anxiety, phobic states.
 9. A method according to claim 1 which isa method for the treatment of depression or depressive disordersincluding major depression, dysthymia, bipolar an dunipolar depression;neurologic or neurodegenerative diseases (including memory loss or othermemory disorders, dementias, Alzheimer's disease).
 10. A methodaccording to claim 1 which is a method for the treatment of disorders oflearning, attention and motivation including Attention DeficitHyperacitvity Disorder, Tourette's disease, impulsivity, antisocial andpersonality disorders, negative symptoms of psyochoses including thosedue to schizophrenia, acquired brain damage and frontal lobe lesions).11. A method according to claim 1 which is a method for the treatment ofhair loss including drug, alcohol and nicotine addiction.
 12. A methodaccording to claim 1 which is a method for the treatment of neurologicor neurodegenerative diseases including memory loss or other memorydisorders, dementias, Alzheimer's disease.
 13. A method according toclaim 1 which is a method for the treatment of movement and locomotordisorders including Parkinson's disease, Huntington's disease, and motordeficits after stoke, head injury, surgery, tumour or spinal cordinjury.
 14. A method according to claim 1 which is a method for thetreatment of diseases related to uncontrolled or abnormal collagen orfibronectin formation including fibrosis of lung, heart andcardiovascular system, kidney and genitourinary tract, gastrointestinalsystem, cutaneous, rheumatologic and hepatobiliary systems.
 15. A methodaccording to claim 1 which is a method for the treatment of delayedonset of labour, impaired cervical ripening, and prevention of prolongedlabour due to fetal dystocia.
 16. A method according to claim 1 which isa method for the treatment of endocrine disorders including adrenal,ovarian and testicular disorders related to steroid or peptide hormoneproduction.
 17. A method according to claim 1 which is a method for thetreatment of breast disorders including fibrocystic disease, impairedlactation, and cancer.
 18. A method according to claim 1 which is amethod for the treatment of immunological disorders including immunedeficiency states, haematological and reticuloendothelial malignancy.19. A method according to claim 1 which is a method for the treatment ofendometrial disorders including infertility due to impairedimplantation.
 20. A method according to claim 1 which is a method forthe treatment of endocrine disorders including adrenal disorders,ovarian disorders, and testicular disorders related to steroid orpeptide hormone production.
 21. A method according to claim 1 which is amethod for the treatment of diseases associated with water balanceincluding impaired or inappropriate secretion of vasopressin.
 22. Amethod according to claim 1 which is a method for the treatment ofplacental insufficiency. 23-44. Canceled.